There are many medical conditions, for example hemorrhagic hypotension and anaphylactic shock, in which significant blood loss and/or hypotension (abnormally low blood pressure) occur leading to reduced tissue oxygenation. For patients with such medical conditions, it is desirable and often critical for their survival to stabilize their blood pressure and to increase the amount of oxygen provided to body tissues by their circulatory systems.
Considerable effort has therefore been expended in developing colloidal substances which may be used as resuscitation fluids and/or blood plasma expanders for stabilizing blood pressure by hemodilution (i.e., increasing blood plasma volume) and which are capable of carrying and delivering oxygen to bodily tissues. The costs, risks (including contamination with disease-causing viruses) and histocompatibility requirements associated with the transfusion of whole blood or blood fractions have stimulated researchers to develop alternate oxygen-carrying substances.
Hemoglobin, the natural respiratory protein of erythrocyte which carries oxygen to body tissues from the lungs, is a potential alternate oxygen-carrying biocolloid. Erythrocytes contain approximately 34 grams of hemoglobin per 100 ml of red cells.
Hemodilution experiments with hemoglobin have revealed that unlike hemodilution with albumin, hemodilution with hemoglobin does not augment cardiac output (whole-body blood flow). During hemodilution with inert colloids such as albumin, whole-body blood flow increases inversely proportional to the level of hemodilution. In other words, an .apprxeq.50% hemodilution (i.e., an .apprxeq.50% decrease in blood red cell mass) increases blood flow .apprxeq.100%, and so on. The physical basis of the hematocrit-blood flow inverse relationship is analogous to viscosity-flow mechanics. In other words, decreases in hematocrit cause decreases in blood viscosity which results in increases in blood flow. Hemoglobin, on the other hand, is not inert. Hemodilution with hemoglobin causes vasoconstriction which results in smaller diameter blood vessels. Therefore, even though blood viscosity is decreased during hemodilution with hemoglobin, the smaller diameter blood vessels resist increases in blood flow. This is analogous to direct mechanical relationship between flow and tube diameter, i.e., decreased diameter results in decreased flow. Whole-body flow (cardiac output) is not increased during hemodilution with hemoglobin because flow of the less viscous blood is opposed by the hemoglobin-mediated decrease in blood vessel diameter.
Increased cardiac output is desirable to increase oxygen delivery to the tissues. Examples of publications describing the lack of increased cardiac output during hemodilution with natural (unmodified) hemoglobin are Sunder-Plassmann et al., Eur. J. Int. Care Med. 1, 37-42 (1975) and Moss et al., Surg. Gyn. Ob., 142, 357-362 (1976).
Hemodilution with modified hemoglobin that has been polymerized also has failed to increase cardiac output. See Vlahakes et al., J. Thorac. Cardiovasc. Surg., 100, 379-388 (1990) which describes the hemodilution of sheep with polymerized bovine hemoglobin prepared by Biopure Corporation; Rausch et al., supra (assigned to Biopure Corporation), which describes similar experiments; and Gould et al., Ann. Surg., 211, 394-398 (1990) and Hobbhahn et al., Acta Anaesthesiol. Scand., 29, 537-543 (1985) which describe hemodiluting baboons and dogs, respectively, with polymerized human hemoglobin solutions. Thus, hemodilution with both natural and modified hemoglobin has failed to increase cardiac output.
Because cardiac output does not increase upon dilution of the blood with hemoglobin, body tissues are required, as one compensatory mechanism, to extract more oxygen from the diluted blood to prevent tissue damage from hypoxia. However, such compensatory mechanisms have real limits in vivo. The heart, for instance, normally functions at about 95% maximal oxygen extraction levels and thus is only capable of increasing oxygen extraction about 5% (assuming 100% efficiency is possible). In the case of hemodilution with albumin, even though arterial oxygen content is decreased, oxygen delivery is essentially maintained at baseline levels because of low viscosity blood that causes flow (cardiac output) to increase proportionally. Therefore, hemodilution with hemoglobin offers no physiological or clinical advantage over hemodiluting with albumin. In fact, oxygen delivery is suboptimal during hemodilution with hemoglobin. This leads us to the central idea of the invention. If cardiac output or whole-body flow were allowed to increase as blood was diluted with hemoglobin then oxygen delivery would be clinically superior to hemodilution with albumin because of the added arterial oxygen content provided by the plasma hemoglobin colloid.
The mechanism of unchanged cardiac output during hemodilution with hemoglobin may be caused by an inactivation of endogenous nitric oxide (NO), also called endothelium-derived relaxing factor (EDRF), which is an important regulator of blood vessel diameter. For nearly 100 hundred years, it has been known that hemoglobin inactivates NO that has been diffused to the blood via the lungs. Only recently has it been discovered that NO also forms biochemically in vivo. There is, however, uncertainty as to whether oxyHb directly reacts with NO or whether an indirect oxyHb-mediated product such as superoxide is responsible. An overview of the possible molecular interactions of oxyHb with NO and relevant compounds is give below.
Initially, the reaction equations between NO and oxyHb/deoxyHb are simple yielding EQU oxyHb+NO.fwdarw.metHb+NO.sub.3.sup.- EQU deoxyHb+NO.fwdarw.NOHb
where metHb is ferric(Fe.sup.3+)hemoglobin, NO.sub.3.sup.- is nitrate and NOHb is nitrosylhemoglobin. However, NO.sub.3.sup.- can react with deoxyHb to yield: EQU 2deoxyHb+NO.sub.3.sup.- +H.sub.2 O.fwdarw.2metHb+NO.sub.2.sup.- +2OH.sup.-
and nitrite (NO.sub.2.sup.-) can react with oxyHb or deoxyHb to yield: EQU 2oxyHb+NO.sub.2.sup.- +H.sub.2 O.fwdarw.2metHb+NO.sub.3.sup.- +2OH.sub.2.sup.- EQU oxyHb+NO.sub.2.sup.- +2H.sup.+.fwdarw.metHb+NO.sub.2 +H.sub.2 O.sub.2 EQU deoxyHb+NO.sub.2.sup.- +H.sub.2 O.rarw..fwdarw.1/2[metHb+NO]+1/2[NOHb]+2OH.sup.-1
where NO.sub.2 is nitrogen dioxide and H.sub.2 O.sub.2 is hydrogen peroxide. The free energy of activation (.DELTA.G) of the last equation is rather low (-21.23 kJ/mole) and therefore metHb . . . NO can be reduced to deoxyHb. However, in vivo experiments have shown that nitrite exposure results in about an equal amount of NOHb and metHb formed in the blood. Furthermore, the .DELTA.G of equation 2 is low (-46.02 Kj/mole) and in the presence of O.sub.2 will proceed according to the .DELTA.G of equation 1 (.apprxeq.-170 Kj/mole). Finally, metHb and H.sub.2 O.sub.2 can react: EQU metHb+H.sub.2 O.sub.2.fwdarw.ferrylhemoglobin+H.sub.2 O
to produce a spectrophotometrically detectable red compound known as ferryl(Fe.sup.++++)hemoglobin. In many of the above reactions, heme or chelatable iron can be substituted for Hb.
Under physiological conditions, i.e., in an oxygenated and heated aqueous with a pH of about 7.4, NO is rapidly converted to nitrogen dioxide: EQU 2NO+O.sub.2.fwdarw.2NO.sub.2
Nitrogen dioxide is quite reactive, and in aqueous solution disproportionates to nitrate and nitrite as: EQU 2NO.sub.2.fwdarw.N.sub.2 O.sub.4 +H.sub.2 O.fwdarw.NO.sub.3.sup.- +NO.sub.2.sup.- +2H.sup.+
The decomposition of NO can also occur via: EQU NO.sub.2 +NO.fwdarw.N.sub.2 O.sub.3 +H.sub.2 O.fwdarw.2NO.sub.2.sup.- +2H.sup.+
The abnormally rapid oxidation of oxyHb by NO is consistent with oxyHb serving as a superoxide (O.sub.2.sup.-) donor, where EQU oxyHb+NO.fwdarw.metHb+ONOO.sup.-.fwdarw.NO.sub.3.sup.-
or as recent studies propose, that NO is inactivated by other sources of O.sub.2.sup.- yielding: EQU NO+O.sub.2.sup.-.fwdarw.ONOO.sup.- +H.sup.+.fwdarw.HO.+NO.sub.2.fwdarw.NO.sub.3.sup.- +H.sup.+
where ONOO.sup.- is peroxynitrite. Hydroxyl radical (HO.) can further react with NO.sub.2.sup.- to form NO.sub.2. Peroxynitrate (O.sub.2 NOO.sup.-) and ONOO.sup.- are also suspected intermediates in the autocatalytic oxidation of oxyHb by NO.sub.2 and NO, respectively.
Considering the autoxidation of oxyHb: EQU oxyHb.rarw..fwdarw.metHb+O.sub.2.sup.- EQU O.sub.2.sup.- +oxyHb.fwdarw.metHb+O.sub.2 +H.sub.2 O.sub.2
These last two equations are slow (the rate constants are 4-6.times.10.sup.3 M.sup.-1 sec.sup.-1). A currently-popular hypothesis is that O.sub.2.sup.- is converted in the presence of iron to the highly toxic HO. radical via the superoxide driven Fenton reaction: EQU O.sub.2.sup.- +Fe.sup.3+.fwdarw.Fe.sup.++ +O.sub.2 EQU 2O.sub.2.sup.- +2H.sup.+.fwdarw.H.sub.2 O.sub.2 +O.sub.2 EQU H.sub.2 O.sub.2 +Fe.sup.++.fwdarw.HO.+OH.sup.- +Fe.sup.3+
Whether oxyHb+H.sub.2 O.sub.2 forms HO. has not been proven. Furthermore, ferric iron (Fe.sup.3+) is sparingly soluble under physiological conditions and, therefore, must be chelated to heme, ferritin, etc to remain in solution. As with metHb, heme-Fe.sup.3+ may also react with H.sub.2 O.sub.2 to form the ferryl-heme radical (.Fe.sup.4+ -heme).
Finally, rather than binding to heme, inactivation of endogenous NO may occur via its reaction with the thiol groups of oxyHb. Recently, it was shown that NO circulates in mammalian plasma primarily as an S-nitroso adduct on the thiol groups of serum albumin.
The biochemical pathway/effect of NO production and metabolism is believed to comprise the following, L-arginine.fwdarw.NO synthase.fwdarw.NO.fwdarw.guanylate cyclase.fwdarw.cyclic GMP.fwdarw.decreased blood vessel diameter. Conceivably, hemoglobin could decrease blood vessel diameter by inactivating any point of the pathway. Furthermore, hemoglobin does not effect the diameter of veins and arteries equally. Equal effect on these two vascular systems is necessary to increase blood flow or cardiac output as observed during albumin hemodilution. Therefore, two mechanisms must be synchronized in order for cardiac output (and oxygenation) to be maximized during hemodilution with hemoglobin: 1) molecular intervention of in vivo hemoglobin chemistry and 2) hemodynamic responsiveness in venous and arterial circulations.
There thus continues to exist a need in the art for new methods, and compositions, useful for hemodilution with hemoglobin which increase cardiac output (oxygen delivery) and give this colloid a clinical advantage over non-oxygenated colloids such albumin.